Omeprazole, which is a proton pump inhibitor, is known as a therapeutic agent of indigestion, stomach ulcer, gastroesophageal reflux disease and laryngopharyngeal reflux disease. The omeprazole, which is a racemate, contains S and R enantiomers at a ratio of 50:50. Both enantiomers in the above acidic condition are converted into an achiral compound and reacted with a cysteine group of H+/K+ATPase to inhibit stomach acid production in a parietal cell of the stomach. The omeprazole and enantiomers are metabolized by CYP2C19 and CYP3A4 which are cytochrome P450 present in a human liver, and main metabolites thereof include 5′-O-desmethyl omeprazole, 5′- and 3′-hydroxyomeprazole and omeprazole sulfone (see Renberg et al., Drug Metab Dispos 17:69-76, 1989; Andersson et al., Clin Pharmacokinet 40:411-426, 2001, Li et al., J Pharmacol Exp Ther 315:777-787, 2005). It has been reported that the R enantiomer is generally metabolized to be 5′-O-desmethyl omeprazole, 5′-hydroxyomeprazole by CYP2C19 and the S enantiomer is generally metabolized to be omeprazole sulfone, 3′-hydroxyomeprazole by CYP3A4.
Cytochrome P450 (P450 or CYP) enzyme is a large family consisting of enzymes serving as catalysts of significantly various oxidation reactions throughout the nature ranging from archaea to bacteria, fungi, plants, animals and human. Due to variety of catalytic function, and a wide range of substrates thereof, P450s are largely useful as a biological catalyst in production of fine chemicals including medical supplies, and the like (see Guengerich, Nat Rev Drug Discov 1:359-366, 2002; Urlacher et al., Trends Biotechnol 24:324-330, 2006; Yun C H et al., Trends Biotechnol 25:289-298, 2007; Lamb et al., Curr Opin Biotechnol 18:504-512, 2007). However, despite of potential usability of the cytochrome P450 enzymes of a mammal in various biotechnological fields as described above, P450s have low stability, catalytic activity, and availability, and thus, are not appropriate as a biological catalyst.
When a prodrug is converted into a biologically “active metabolite” by P450s by humans during development of the drug (see Johnson et al., Breast Cancer Res. Treat 85:151-159, 2004), a large amount of pure metabolites are required for a research of efficacy, toxicity, pharmacokinetics, and the like, of the drug. In addition, when the metabolite itself has a biological activity, direct administration of the metabolite in vivo has a large benefit, and thus, mass-production of the metabolite is important.
When the omeprazole is administered into a human body, since the omeprazole is metabolized by CYP2C19 and CYP3A4, a rate at which the metabolite is produced may vary depending on the degree of expression of the enzymes. In addition, a drug interaction problem with other drugs metabolized by the enzymes occurs. Therefore, when the omeprazole metabolite is directly used as a drug, the drug interaction problem may be avoided.
However, since there are various problems in chemically synthesizing pure metabolites, in order to product a metabolite of a drug or a drug candidate as an alternative of the metabolite chemical synthesis, P450 is used. The production of the metabolites using human P450s expressed from E. coli (see Yun et al., Curr Drug Metab 7:411-429, 2006) or insect cells (see Rushmore et al., Metab Eng 2:115-125, 2000; Vail et al., J Ind Microbiol Biotechnol 32:67-74, 2005) has been reported. However, these systems have problems such as expensive cost and low productivity due to limited stability, slow reaction rate, and the like (see Guengerich et al., Crit Rev Toxicol 26:551-583, 1996). Accordingly, a method for using engineered bacterial P450 enzymes having a desired catalytic activity as an alternative for producing metabolites in human has been suggested (see Yun C H et al., Trends Biotechnol 25:289-298, 2007).
Meanwhile, heme domain of P450 BM3 (CYP102A1) derived from Bacillus megaterium has a mono oxygenase activity, which is significantly similar to a member of mammalian of CYP4A (fatty acid hydroxylase) family. Naturally, it is formed of single polypeptides in which a CYP102A1 reductase domain having a mammal-like diflavin reductase function is fused to a C-terminal of the P450 heme domain. The fusion of two enzyme activities makes a fusible CYP102A1 to be a desirable mammal model, in particular, a desirable model of a human P450 enzyme. It has been reported that CYP102A1 mutants genetically engineered through logical design or directed evolution oxidize several substrates of human P450 to product a metabolite having higher activity (see Kim et al., Drug Metab Dispos 36:2166-2170, 2008, Kim et al., Drug Metab Dispos 37:932-936, 2009, Kim et al., J Mol Catal B: Enzym 63:179-187, 2010; Otey et al., Biotechnol Bioeng 93:494-499, 2006; Yun C H et al., Trends Biotechnol 25:289-298, 2007).
Based on the above-description, it has been suggested that the mutants of CYP102A1 may be developed as a biological catalyst for detection and synthesis of the drug. Recently, it has been reported that several selected mutants may allow the CYP102A1 enzyme to product a metabolite in human as a drug (see Kim et al., Drug Metab Dispos 36:2166-2170, 2008, Kim et al., Drug Metab Dispos 37:932-936, 2009); however, a method for biologically producing a metabolite in human from the omeprazole has not been reported yet.